Rapid expression of ornithine decarboxylase mRNA in a macrophage-like cell line: cAMP repression of the requirement for prior protein synthesis.
Rapid expression of ornithine decarboxylase mRNA in a macrophage-like cell line: cAMP repression of the requirement for prior protein synthesis.
复制标题
鸟氨酸脱羧酶 mRNA 在巨噬细胞样细胞系中的快速表达：cAMP 抑制先前蛋白质合成的需要。
DOI：
10.1002/jcp.1041340317
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发表时间：
1988
影响因子：
5.6
通讯作者：
Taffet,SM
中科院分区：
文献类型：
--
作者：
Shurtleff,SA;McElwain,CM;Taffet,SM
用细菌脂多糖（LPS）处理后，在RAW 264巨噬细胞样细胞系中迅速诱导鸟氨酸脱羧酶（ODC）活性。用32 P标记的cDNA探针进行北方印迹和斑点印迹分析。用1.0 μg/ml LPS刺激RAW 264细胞后1小时内ODC mRNA表达增加。对分离的细胞核的转录速率分析表明，转录速率的增加有助于ODC mRNA的增加。ODC mRNA水平持续上升4小时，达到基础水平的8倍。在刺激前，ODC mRNA显示为单一的2.2 kb条带。刺激后，2.2 kb条带增强，并通过北方凝胶分析观察到第二条（2.7 kb）条带。当使用12-O-十四酰基-佛波醇-13-乙酸酯（TPA）作为刺激物时，证明了类似的诱导。通过向细胞孵育混合物中加入放线菌酮（25 μg/ml）或茴香霉素（0.1 mM）来阻断LPS或TPA对ODC mRNA的诱导。这表明蛋白质合成是LPS或TPA诱导ODC mRNA的先决条件。在LPS处理后延迟放线菌酮添加的实验表明，在前30分钟内发生了一些所需的蛋白质合成，并且如果在加入放线菌酮之前蛋白质合成持续至少2小时，则ODC mRNA的完全表达是可能的。已经显示，除了LPS之外，用8-溴-cAMP刺激与单独的LPS或TPA诱导相比增强了ODC的诱导。用LPS和cAMP或TPA和cAMP的组合刺激处理后，用放线菌酮或茴香霉素阻断ODC mRNA的诱导是不可能的，这表明当cAMP用作共诱导剂时，不需要蛋白质合成。因此，我们已经表明，在同一细胞中，ODC mRNA可以通过两种不同的途径诱导，一种需要蛋白质合成，一种不需要蛋白质合成。
Ornithine decarboxylase (ODC) activity was rapidly induced in the RAW264 marcophage‐like cell line after treatment with bacterial lipopolysaccharide (LPS). ODC mRNA leveis were determined by isolating cellular RNA, followed by Northern blot and dot blot analysis using a32P‐labeled cDNA probe. ODC mRNA levels increased within 1 hour of stimulation of RAW264 cells with 1.0 μg/ml LPS. Transcription rate analysis on isolated nuclei indicated that an increase in transcription rate contributed to this increase in ODC mRNA. ODC mRNA levels continued to rise for 4 hours, peaking at eight times the basal level. ODC mRNA appeared as a single 2.2‐kb band prior to stimulation. After stimulation, the 2.2‐kb band intensified, and a second (2.7 kb) band was seen by Northern gel analysis. Similar induction was demonstrated when 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) was used as the stimulus. The induction of ODC mRNA by either LPS or TPA was blocked by the addition of cycloheximide (25 μg/ml) or anisomycin (0.1 mM) to the cellular incubation mixture. This indicated that protein synthesis was required as a prerequisite to LPS or TPA induction of ODC mRNA. Experiments in which cycloheximide addition was delayed after LPS treatment indicated that some of the required protein synthesis occurred within the first 30 minutes and that complete expression of ODC mRNA was possible if protein synthesis continued for at least 2 hours before cycloheximide was added. Stimulation with 8‐bromo‐cAMP in addition to LPS has been shown to enhance the induction of ODC over that induced by LPS or TPA alone. It was not possible to block ODC mRNA induction with cycloheximide or anisomycin after treatment with the combined stimulus of LPS and cAMP or TPA and cAMP, indicating that protein synthesis was not required when cAMP was used as a coinducer. Thus, we have shown that in the same cell, ODC mRNA can be induced by two different pathways, one requiring protein synthesis and one not requiring protein synthesis.